Method for characterizing size and composition of nanoparticles using asymmetrical flow field-flow fractionation combined with inductively coupled plasma optical emission spectrometry

Abstract

A method for characterizing the size and composition of nanoparticles using asymmetrical flow field-flow fractionation combined with inductively coupled plasma optical emission spectrometry, comprising: S1, performing an anaerobic pretreatment on an asymmetrical flow field-flow fractionation instrument; and S2, loading a sulfate solution treated with sulfate-reducing bacteria into the asymmetrical flow field-flow fractionation instrument, and obtaining characterization results after an injection stage, an elution stage, and a flushing stage. The sulfate-reducing bacteria are used in combination with the asymmetrical flow field-flow fractionation instrument to detect the sulfate solution, and the sulfate reducing bacteria are used to reduce sulfate into sulfide, thereby enabling effective detection of sulfate.

Description

A method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry. Technical Field

[0001] This invention relates to a method of using an asymmetric flow field instrument, and more particularly to a method for characterizing the particle size and composition of nanoparticles using inductively coupled plasma atomic emission spectrometry (ICP-AES) with asymmetric flow-field coupling. Background Technology

[0002] Currently, there is no method capable of simultaneously separating and qualitatively and quantitatively characterizing the particle size distribution and composition of nanoparticles. Therefore, this method is compared with existing nanoparticle characterization methods. Existing nanoparticle characterization methods include: transmission electron microscopy (TEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and inductively coupled plasma optical emission spectrometry (ICP-OES), etc.

[0003] Transmission electron microscopy (TEM) possesses atomic-level resolution, enabling the observation of not only the microscopic morphology of samples but also the characterization of the internal structure of the observed region. The principle of TEM is as follows: a high-voltage electron source releases high-speed electrons, which are then condensed into a parallel electron beam by a condenser lens system. This beam is incident on and passes through a localized region of the sample. The electron beam interacts with nanoparticles, modulating the phase and amplitude of the incident electron beam to form an outgoing electron beam. This outgoing electron beam passes through the objective lens system, converging on the focal plane of the objective lens to form a diffraction spot. It then propagates further downwards to the image plane of the objective lens, forming the first magnified image of the sample. TEM allows the study of size, shape, and crystal structure at the single-particle level.

[0004] Dynamic light scattering (DLS) is a technique that derives particle size information from samples by measuring the fluctuations in the intensity of scattered light. Because molecules in the sample are constantly undergoing Brownian motion, this motion causes changes in the scattered light. By utilizing the fluctuations in scattered light over time, a relevant equation is derived, leading to the average velocity (Dt) of the particles in the sample, and ultimately, the average hydrodynamic diameter. DLS offers advantages in particle size detection due to its speed and simplicity, and it has statistical significance. However, because the intensity of scattered light is proportional to the sixth power of the particle size, larger particles are scattered more strongly, thus the average particle size result for widely distributed samples tends to favor larger particles. Therefore, DLS is suitable for particle size detection in monodisperse systems with relatively narrow distributions. Furthermore, DLS experiments are susceptible to the effects of dust or impurities, making sample filtration and centrifugation crucial.

[0005] Nanoparticle tracking analysis involves irradiating a solution of suspended particles with a concentrated laser beam through a glass prism. A chrome-plated glass surface minimizes background signals, allowing the intensity of scattered light from each particle to be detected. This enables observation of the Brownian motion of the nanoparticles in the solution and image capture. By tracking and analyzing the Brownian motion of the particles, the particle size is calculated using the Stokes-Einstein equation, and the concentration is derived from the number of particles. Nanoparticle tracking analysis offers higher accuracy than dynamic light scattering analysis. However, like dynamic light scattering, the signals from particles can be blocked, leading to a bias towards larger particles. Furthermore, the limited focal plane of the image means that the three-dimensional motion of the particles is ultimately represented as a two-dimensional trajectory on the instrument, resulting in some degree of inaccuracy in the final results.

[0006] Inductively coupled plasma atomic emission spectrometry (ICP-OES) utilizes the high temperature generated by ICP plasma to atomize or ionize the analyte, creating an excited state that produces a characteristic emission spectrum. By detecting the wavelength and intensity of the spectral lines, the presence and concentration of the analyte in the sample can be determined. ICP-OES can simultaneously determine most metallic and non-metallic elements, such as sulfur, and boasts advantages such as low detection limits and high precision.

[0007] Current transmission electron microscopy (TEM) methods suffer from limitations. The limited amount of nanoparticles used in electron microscopy can lead to a lack of overall statistical significance in the measurement results. Furthermore, the high surface activity and tendency of nanoparticles to aggregate necessitate ultrasonic dispersion before sample preparation. Accurate results are also difficult to obtain for nanoparticle samples that are susceptible to strong electron beam bombardment. Additionally, the nanoparticles must be electronically transparent and able to withstand the high vacuum and beam energy used in the characterization process. Sample damage is particularly common due to the presence of high-energy electron beams.

[0008] The existing dynamic light scattering method, because the intensity of the scattered light is proportional to the sixth power of the particle size, has a stronger scattering effect on large particles. This results in an average particle size result that is biased towards large particles for samples with a wide distribution. The result may not be accurate or representative enough, and it is easily affected by dust or impurities, which will increase the error of the result and increase the complexity and difficulty of operation.

[0009] Existing nanoparticle tracking analysis methods suffer from light scattering by particles, which causes signals between particles to be blocked, resulting in a bias towards larger particles in the obtained particle size. In addition, due to the limitation of the limited focal plane of the imaging, the three-dimensional motion of the particles is actually presented as a two-dimensional motion trajectory on the instrument, leading to a certain deviation in the final result.

[0010] Existing inductively coupled plasma atomic emission spectrometry (ICP-AES) suffers from complex spectral lines, significant spectral interference, and requires large sample quantities. Furthermore, when used alone, it necessitates pretreatment such as acidification, which can damage the sample structure.

[0011] To overcome the shortcomings of existing technologies, such as complex operation, over-reliance on separation and extraction techniques, and relatively limited characterization capabilities, and to achieve efficient separation and characterization of nanoparticles, this method uses an asymmetric flow field analyzer (AF4, hereinafter referred to as the flow field analyzer) coupled with ICP-OES to separate and characterize nanoparticles. This method has the advantages of convenient operation, simultaneous determination of multiple elements, simple pretreatment, low destructiveness, and low detection limit. Summary of the Invention

[0012] This invention provides a method for characterizing the particle size and composition of nanoparticles using inductively coupled plasma atomic emission spectrometry (ICP-AES) with asymmetric flow-field coupling, in order to detect and characterize sulfate solutions treated with sulfate-reducing bacteria.

[0013] This invention provides a method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry, comprising:

[0014] S1. Pre-treat the asymmetric flow field instrument in an oxygen-free environment;

[0015] S2. The sulfate solution treated with sulfate-reducing bacteria was loaded into an asymmetric flow field analyzer and subjected to a sample injection stage, a flushing stage, and a rinsing stage to obtain the characterization results.

[0016] Furthermore, the sulfate solution treated with sulfate-reducing bacteria also contains metal ions, specifically Fe. 2+ Fe 3+ Cu 2+ Al 3+ One or more of them.

[0017] Furthermore, the concentration of the metal ions is 100-400 μmol·L⁻¹. -1 .

[0018] Furthermore, the method for preparing the sulfate solution after treatment with sulfate-reducing bacteria is as follows:

[0019] S101. Take a sulfate solution;

[0020] S102. Prepare bacterial suspension with OD600=0.8 using anaerobic background solution. Mix the bacterial suspension and Cu stock solution at a volume ratio of 1:1 and react with horizontal shaking for 24 h.

[0021] S103. Take the solution after S102 treatment and centrifuge it to obtain the solution to be tested.

[0022] Furthermore, the anaerobic background solution is a 1 mmol / L Na2SO4 anaerobic background solution, and the process is carried out at a temperature of 30±1℃.

[0023] Furthermore, the horizontal oscillating reaction is specifically carried out at 30±1℃ and 150rpm for 24h.

[0024] Further, S1. pre-treating the asymmetric flow field instrument in an oxygen-free environment includes:

[0025] Prepare a 25 μM NaCl solution with oxygen-free water, select a low flow rate of 0.05 mL / min, and control the operation of the asymmetric flow field analyzer for 12-36 hours to ensure that the asymmetric flow field analyzer is in an anaerobic environment.

[0026] Furthermore, in step S2, the sulfate solution treated with sulfate-reducing bacteria is loaded into an asymmetric flow field analyzer, and after the sample introduction stage, rinsing stage, and flushing stage, the characterization results are obtained, including:

[0027] S201. In the anaerobic workstation, take 2.5 mL of sulfate solution after 24 h of reaction treated with sulfate-reducing bacteria and put it into a 1 mL sample bottle for determination;

[0028] S202. After the sample introduction stage, flushing stage, and rinsing stage, the characterization results are obtained. Further, the sample introduction stage includes: taking the sulfate solution treated with sulfate-reducing bacteria from the sample bottle, setting the asymmetric flow field analyzer program to a detector flow rate of 0.5 mL / min; during focusing: sample introduction speed: 0.20 mL / min, sample introduction time: 5 min, sample volume: 200 μL, lateral flow rate: 1.00 mL / min, focusing pump: 1.3 mL / min.

[0029] Furthermore, the elution stage includes setting the elution process conditions as follows: (1) lateral flow rate 1.00 mL / min, mode constant, time 15 min; (2) lateral flow rate 1.00 mL / min, mode Power, Exponent 0.20, time 30 min; (3) lateral flow rate 0.05 mL / min, mode Power, Exponent 0.80, time 10 min; (4) lateral flow rate 0.00 mL / min, mode constant, time 30 min.

[0030] Compared with the prior art, the present invention utilizes sulfate-reducing bacteria in conjunction with an asymmetric flow field instrument to detect sulfate solutions. The sulfate-reducing bacteria convert sulfate ions into sulfur (S), thereby achieving effective detection of sulfate ions. Attached Figure Description

[0031] Figure 1 shows the AF4-UV results of nanospheres of different sizes in the embodiments of the present invention;

[0032] Figure 2 shows the elemental content in MNPs determined by AF4 coupled with ICP-OES in an embodiment of the present invention. The vertical axis represents the emission spectrum signal intensity value (cps); where (a) Cu; (b) Fe; (c) S; (d) Mn. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0034] The asymmetric flow field analyzer (Poatnova, AF2000MT, GERMAN), hereinafter referred to as the flow field analyzer, involves asymmetric flow fields in separation. Under the influence of asymmetric flow field forces and opposing diffusion fields, the sample forms different equilibrium layers within the channel. During the sample introduction stage, before separation begins, the sample enters the channel from the inlet and is subjected to a unidirectional cross-flow force from the top of the channel. The pressure generated by the cross-flow causes the colloidal particles of the sample to be retained on a dialysis membrane that only allows small molecule solutes to pass through. At this point, the sample diffuses in the vertical direction of the channel, and the diffusion coefficient depends on factors such as the size and density of different colloidal particles. Colloidal particles with larger particle sizes or densities have smaller diffusion coefficients and aggregate near the lower part of the channel near the membrane, while particles with smaller particle sizes or densities tend to be closer to the middle of the channel space. Simultaneously, the sample is also subjected to a focusing flow field force opposite to the elution direction. Under the combined action of the channel flow and the focusing flow, the sample is concentrated near the inlet, and only small molecule solutes can be eluted through the dialysis membrane. After sample introduction, the focusing flow velocity returns to zero, and the colloidal particles are eluted by the channel flow and then flow towards the detector. According to boundary layer theory, within the same cross-section of the channel, the fluid velocity near the boundary is slower, while the fluid velocity near the center of the channel is faster, exhibiting a parabolic streamline along the cross-section. Therefore, smaller particles are flushed out first by the faster fluid and preferentially enter the detector, while larger particles are gradually flushed out as the cross-flow decreases.

[0035] The field flow meter can be coupled with many detectors, such as ultraviolet-visible spectrophotometer (UV-vis), multi-angle static laser particle size analyzer (MALS), fluorescence detector, differential detector, ICP-OES, etc. By analyzing different characteristics of colloidal particles through different detectors, it is possible to simultaneously detect, separate and characterize colloidal particles online.

[0036] By using a combined field flow analyzer and ICP-OES, nanoparticles of different sizes were separated and their elemental composition and particle size were characterized. Taking advantage of the ability of ICP-OES to simultaneously determine most metallic and non-metallic elements such as sulfur, it is possible not only to explore the relationship between the particle size and elemental composition of nanoparticles, but also to determine the binding of nanoparticles. This has important reference value for the study of the environmental effects and biotoxicology of nanoparticles at the nanoscale.

[0037] To further illustrate the solution of the present invention, specific embodiments are disclosed below.

[0038] The experiment used sulfate-reducing bacteria (SRB) and Cu 2+The interaction is complex. SRB is a common characteristic microbial community in sulfur-rich anaerobic sedimentary habitats in mining areas. It can generate sulfides through sulfate reduction reactions, which bind with heavy metal ions to mediate the formation of metal-bearing nanoparticles (MNPs). Using asymmetric flow field separation and characterization techniques, the binding of nanoparticles can be determined by characterizing the Cu, Fe, Mn, and S elements.

[0039] The reaction was carried out under anaerobic background conditions of Na₂SO₄ at pH 6 and an ionic strength of 1 mmol / L, at a temperature of 30 ± 1 °C. First, a bacterial culture with an OD₆₀ = 0.8 was prepared using the background solution. 20 mL of the bacterial culture was added to a 50 mL centrifuge tube, followed by 20 mL of Cu stock solution (specifically, a 200 μmol / L Cu(NO₃)₂ solution), bringing the copper concentration in the system to 200 μmol / L and the total solution volume to 40 mL. The reaction was carried out at 30 ± 1 °C with constant temperature shaking at 150 rpm for 24 h. After the reaction, 5 mL was transferred to a 3 kDa (approximately 1 nm) ultrafiltration centrifuge tube (Microsep Advance, Pall) for centrifugation to ensure the nanoparticles and dissolved Cu were properly separated. 2+ Complete separation of metal ions.

[0040] Before separation, a 25 μM NaCl background solution was prepared using oxygen-free water and run overnight at a low flow rate of 0.05 mL / min to ensure an anaerobic environment during separation. In the anaerobic workstation, 2.5 mL of the sample after 24 hours of reaction was transferred to a 1 mL vial supplied with AF4 for analysis. The prepared SRB and Cu were then... 2+ The products resulting from the interaction were separated using an asymmetric flow field analyzer (AF2000MT, Postnova, Germany). The operating method was set on the flow field analyzer software, which consisted of three steps: the sample introduction stage, the flushing stage, and the rinsing stage.

[0041] Specific method parameter settings: Detector flow rate 0.5 mL / min. During focusing: Injection rate: 0.20 mL / min, injection time 5 min, injection volume: 200 μL, lateral flow rate: 1.00 mL / min, focusing pump: 1.3 mL / min. Elution process: (1) Lateral flow rate 1.00 mL / min, constant mode, time 15 min. (2) Lateral flow rate 1.00 mL / min, power mode, Exponent 0.20, time 30 min. (3) Lateral flow rate 0.05 mL / min, power mode, Exponent 0.80, time 10 min. (4) Lateral flow rate 0.00 mL / min, constant mode, time 30 min. The total running time for one sample is 90 min.

[0042] The AF4-UV-ICP-OES technique was used to obtain ultraviolet signals and determine the spectral signals of Cu, Fe, Mn, and S elements by combining UV-Vis and ICP-OES, and then MALS was used to obtain particulate signals. Calibration was performed using latex standard samples to obtain the relationship between the appearance time of product signals and particle size, thus revealing the particle size of the separated samples and the distribution of Cu in different particle sizes. The AF4-UV-ICP-OES technique can separate the products resulting from the interaction of SRB and Cu2+ in different states, and the particle size of the products can be determined by the peak times of standards with different particle sizes. ICP-OES was then used to analyze the distribution of Cu, Fe, Mn, and S elements in products of different sizes.

[0043] To perform particle size analysis on the separation results, it is first necessary to investigate the separation characterization of nanosphere standards of different sizes under the parameter settings of this experiment, and obtain the corresponding peak times of the nanosphere standards. This experiment selected nanosphere standards provided by POSTNOVA, Germany, with sizes of 20 nm, 200 nm, 600 nm, and 900 nm. All standards contained trace amounts of surfactant to prevent agglomeration and enhance stability, and were equipped with NIST calibration traceability certificates. The AF4-UV results for nanosphere standards of different sizes are shown in Figure 1. It can be seen that the peak particle size is 20 nm between 1 and 10 min, the peak particle size at 20 min is between 200 nm, the peak at 30 min represents a particle size of 600 nm, and the peak at approximately 60 min is 900 nm. These results will be used for particle size analysis.

[0044] The field flow meter effectively separates nanoparticles of different sizes from other dissolved substances. After ICP-OES detection, it was found that the signal curves of Cu and S are basically consistent, and the signal values ​​show a proportional correlation. The signal curves of Fe and Mn metal ions are only moderately consistent with those of S.

[0045] The first significant signal peak was observed to appear between 25 and 30 min (Figure 2-a, b, d), indicating that Cu formed in the system at this time. x S nanoparticles were also formed, along with a small amount of Mn. x S nanoparticles, with a particle size of 200–600 nm, exhibit a second signal peak at 30–40 min (Figure 2-a, b, d), where Cu and S signals are more significant, while Mn signals are less pronounced. At this point, the nanoparticle size is 600–900 nm. Furthermore, significant Fe ion signal peaks appear around 30 and 50 min (Figure 2-b), indicating the formation of Fe-containing nanoparticles in the system, but not demonstrating the binding of Fe to S. Particularly between 30 and 40 min, the Fe signal is not significant, suggesting that the 600–900 nm nanoparticles formed in the system contain a low proportion of Fe.

[0046] In summary, the present invention has the following beneficial effects:

[0047] 1. For the first time, an asymmetric flow field analyzer combined with inductively coupled plasma atomic emission spectrometry was used to characterize nanoparticles, and good characterization results were obtained. This method can be used to separate and characterize nanoparticles.

[0048] 2. Explore suitable flow metering methods for samples, including axial flow velocity, cross flow velocity, injection time, rinsing time, etc., to improve the elution and detection effects of samples;

[0049] 3. Practice has proven that the method parameters of the asymmetric flow field analyzer are effective for separating colloidal particles and nanoparticles, and the specific settings of the flow field analyzer method parameters should be protected.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.

Claims

1. A method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry, characterized in that, include: S1. Pre-treat the asymmetric flow field instrument in an oxygen-free environment; S2. The sulfate solution treated with sulfate-reducing bacteria was loaded into an asymmetric flow field instrument and subjected to a sample injection stage, a flushing stage, and a rinsing stage to obtain the characterization results.

2. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 1, characterized in that, The sulfate solution treated with sulfate-reducing bacteria also contains metal ions, specifically Fe. 2+ Fe 3+ Cu 2+ Al 3+ One or more of them.

3. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 2, characterized in that, The concentration of the metal ions is 100-400 μmol·L⁻¹. -1 .

4. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 3, characterized in that, The method for preparing the sulfate solution after treatment with sulfate-reducing bacteria is as follows: S101. Use sulfate solution as the anaerobic background solution; S102. Prepare bacterial suspension with OD600 = 0.8 using anaerobic background solution. Mix the bacterial suspension and Cu stock solution at a volume ratio of 1:1 and react with horizontal shaking for 24 h. S103. Take the solution after S102 treatment and centrifuge to obtain the test solution.

5. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 4, characterized in that, The anaerobic background solution was a 1 mmol / L Na2SO4 anaerobic background solution, and the process was carried out at a temperature of 30±1℃.

6. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 4, characterized in that, The horizontal oscillating reaction is specifically carried out at 30±1℃ and 150rpm for 24h.

7. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 1, characterized in that, S1. Pre-treatment of the asymmetric flow field instrument with an anaerobic environment includes: preparing a 25 μM NaCl solution with anoxic water, selecting a low flow rate of 0.05 mL / min, and controlling the operation of the asymmetric flow field instrument for 12-36 h to ensure that the asymmetric flow field instrument is in an anaerobic environment.

8. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 1, characterized in that, S2. The sulfate solution treated with sulfate-reducing bacteria is loaded into an asymmetric flow field analyzer, and after the injection, flushing, and rinsing stages, the characterization results are obtained, including: S201. In the anaerobic workstation, take 2.5 mL of sulfate solution after 24 h of reaction treated with sulfate-reducing bacteria and put it into a 1 mL sample bottle for determination; S202. After the sample introduction stage, flushing stage and rinsing stage, the characterization results are obtained.

9. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 8, characterized in that, The sample introduction stage includes: taking the sulfate solution after treatment with sulfate-reducing bacteria from the sample bottle, and setting the program of the asymmetric flow field instrument to a detector flow rate of 0.5 mL / min; during the focusing process: sample introduction speed: 0.20 mL / min, sample introduction time: 5 min, sample introduction volume: 200 μL, lateral flow rate: 1.00 mL / min, focusing pump: 1.3 mL / min.

10. The method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field coupled inductively coupled plasma atomic emission spectrometry according to claim 9, characterized in that, The elution phase includes setting the elution process conditions as follows: (1) lateral flow rate 1.00 mL / min, mode constant, time 15 min; (2) lateral flow rate 1.00 mL / min, mode Power, Exponent 0.20, time 30 min; (3) lateral flow rate 0.05 mL / min, mode Power, Exponent 0.80, time 10 min; (4) lateral flow rate 0.00 mL / min, mode constant, time 30 min.

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